Human papillomaviruses (HPV) are a family of sexually transmitted DNA viruses with over 100 different genotypes. The genotypes are divided into the low-risk and high-risk categories based on the spectrum of lesions they induce. The low-risk types primarily induce benign genital condylomas and low-grade squamous intraepithelial lesions whereas the high-risk types are associated with the development of anogenital cancers and can be detected in >99% of cervical cancers, with HPV16 found in about 50% of cases. In the United States, an estimated 75% of the sexually active population acquires at least one genital HPV type during their lifetime.
While morbidity and mortality caused by cervical cancer can be reduced with effective Papanicolaou (Pap) smear screening, early detection, and treatment, none of these are readily available in developing countries, the origin of many immigrants to the U.S. In developing countries, cervical cancer remains the second leading cause of cancer-related deaths among women. Importantly, the burden of this disease is expected to increase dramatically in the next decades due to changing demographics. Even in the U.S. where screening programs have reduced the overall rate of invasive cancer, a disparity exists in the incidence of cancer development between white non-Hispanic, black, Hispanic, and economically disadvantaged women. Penetrance of the current FDA-approved preventive vaccine for HPV, GARDASIL® (Merck), in the U.S. has been disappointing. The vaccine was administered to only 25% of girls ages 13-17, 10% of all females ages 18-26, and was given to only 1.1% of Hispanic women in 2007. Moreover, the vaccine is ineffective in women that have already been infected with the virus, whether they have developed (pre-) cancerous cervical lesions or not. Given the lifetime risk of HPV infection and the fact that populations severely underrepresented in vaccine coverage will likely continue to develop cervical and other HPV-related diseases at an alarming rate, it is clear that there is an enormous need for therapeutic approaches that would mitigate carcinogenic effects after viral infection has occurred.
HPV are non-lytic, non-enveloped viruses. Their genome coding regions are denoted E and L for “early” and “late” proteins. The E proteins fulfill regulatory functions vital for genome replication, two of which (E6 and E7) play a significant role in oncogenesis, while the two L proteins (L1 and L2) are the self-assembling capsid proteins responsible for DNA packaging and virion assembly. Infection by papillomaviruses is unique in that its productive lifecycle is coupled to the cellular differentiation of proliferating host epidermal or mucosal basal epithelial cells. HPV remains suprabasal throughout its lifecycle and therefore only contacts cells in the epidermis such as basal cells and Langerhans cells (LC). Due to the coupling of its lifecycle to cellular differentiation, it is difficult to produce HPV virions in vitro. As an alternative to HPV virions, HPV virus-like particles (VLP) and HPV pseudovirions, capable of carrying reporter plasmids, have been developed and are both technologies that are established in the laboratory of Applicants.
Persistence of a high-risk HPV infection is a major risk factor in the development of cervical cancer. While a majority of women infected with HPV clear the virus, the time taken to do so can range from many months to years. About 15% of women that have high-risk HPV infections do not initiate an effective immune response against HPV, allowing the virus to persist for decades. The slow clearance rate and lack of effective immunity indicates that HPV somehow escapes the immune response.
HPV has developed a variety of escape mechanisms that circumvent immediate elimination, allowing viral replication and persistence in the host. Applicants have shown that HPV manipulates LC as a mechanism of immune escape, shown in FIG. 4. LC located in the epithelial layer of the skin and mucosa are the first and critical APC to come into contact with HPV. Consequently, LC are responsible for initiating an effective immune response against HPV infection. Upon recognition of a foreign antigen, LC undergo maturation, which consists of phenotypic and functional changes including up-regulation of co-stimulatory molecules CD80 and CD86, MHC class I and II, chemokine receptors such as CCR7, secretion of cytokines and chemokines, and migration to regional lymph nodes. Applicants have established that LC exposed to HPV16 L1 L2 VLP do not up-regulate co-stimulatory molecules and chemokine receptors, do not secrete cytokines and chemokines and do not initiate epitope-specific immune responses against HPV16 VLP-derived antigens. In contrast, myeloid DC are activated by HPV16 L1 L2 VLP and once activated, stimulate HPV-specific T cells. Different intracellular signaling cascades are initiated in DC versus LC upon uptake of HPV16 L1 L2 VLP. When stimulated with HPV16 L1 L2 VLP, the mitogen-activated protein kinase (MAPK) pathway is activated in DC whereas it is inactivated in LC. However, the phosphoinositide 3-kinase (PI3K) pathway is activated in LC, leading to a signaling cascade that results in the inactivation of Akt. HPV16 E7-specific T cells can recognize and kill LC exposed to HPV16 L1L2-E7 chimeric VLP (cVLP), indicating that HPV peptides are presented by LC after cVLP internalization but that HPV16 L1 L2 VLP inhibit LC from inducing an immune response. Taken together, the data suggest that LC present HPV-derived peptides in the absence of co-stimulation, thereby becoming tolerogenic and immune-suppressive. This in turn can lead to persistence of the HPV infection and an increased likelihood of cancer development.
Prophylactic vaccines for HPV induce high titers of HPV-neutralizing antibodies and have shown high efficacy up to 6.5 years of follow-up and sustained levels of antibodies. However, in women infected with HPV, a phase 3 trial found no evidence of accelerated viral clearance in the vaccinated group as compared to the control group, illustrating conclusively the lack of therapeutic efficacy in preventive VLP-based vaccines. Furthermore, with the long incubation time of HPV, several mechanisms of immune evasion and only a few years of observation, sustained efficacy of prophylactic vaccines on cancer prevention has yet to be determined. The fact that about one third of cervical cancer is caused by HPV types other than those currently present in the vaccines increases the scope of the problem. Thus, it will take decades to be able to detect a quantifiable effect on cervical cancer rates. Meanwhile, the need for therapies to treat HPV infections and associated lesions remains for the hundreds of millions of women worldwide that are currently infected with high-risk HPV or will become infected in the coming years.
Surgery, the standard of care for patients with cervical intraepithelial neoplasia (CIN) lesions, is usually quoted as being up to 90% effective in removing CIN lesions when followed for one year. However, it is less effective when women are monitored over their lifetime. Greater than 80% of women that undergo surgical procedures will subsequently return in need for a second related procedure in cases where surgery does not remove the HPV infection or where elimination of one HPV type encourages re-activation of secondary HPV infections. The therapeutic vaccines that are in development aim to control malignancy by activating the patient's own cellular immune response and target antigens present in the (pre-) cancer cells. Several candidate vaccines have been developed over the last fifteen years; however, to date, there is not a single cancer vaccine that has been approved by the Food and Drug Administration.
Interventions that prevent HPV infections from reaching the stage of inducing carcinogenesis are needed. Such interventions are feasible, since HPV infection can be detected early on with a commercially available HPV detection kit (Digene Corp.).
Several lines of evidence support the importance of the cellular immune system in controlling the pathogenesis of HPV and associated cervical lesions. Firstly, 25-40% of HPV positive, mildly dysplastic lesions resolve spontaneously or shortly after local biopsy suggesting that induction of local inflammation may be involved with regression. Secondly, immunodeficiency is associated with increased incidence of HPV infection. Regressing HPV-associated skin warts and genital warts often have T lymphocytes in the lesions, suggesting that active cell-mediated immune responses to HPV may be a component of regression of the disease. Taken together, these studies illustrate that therapeutic regimens for HPV infection and its associated diseases should aim to induce strong cellular immunity at the site of infection.
Therefore, there is a need for both an effective immunological treatment of HPV over the lifetime of women as well as a method of overcoming the HPV-induced immune suppression of LC that prevents effective treatment.